Walch Phddissert

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SUBFORNICAL ORGAN ANGIOTENSIN TYPE 1 RECEPTOR EXPRESSION

AND DRINKING BEHAVIOR IN BILE DUCT LIGATED RATS

ADISSERTATION

Presented to the F !"#t$ o%

The Un&'ers&t$ o% Te( s He #th S!&en!es Center t S n Anton&o

Gr d" te S!hoo# o% B&o)ed&! # S!&en!es

&n * rt& # %"#%#)ent

o% the re+"&re)ents%or the de,ree o%

DOCTOR OF PHILOSOPHY

B$

-ose*h D. / #!h B.S.

S n Anton&o0 Te( s

-"#$ 20 31


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Co*$r&,ht

B$-ose*h D. / #!h

31A## R&,hts Reser'ed


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SUBFORNICAL ORGAN ANGIOTENSIN TYPE 1 RECEPTOR EXPRESSION ANDDRINKING BEHAVIOR IN BILE DUCT LIGATED ANI4ALS

-ose*h D. / #!h

APPROVED5

-. Tho) s C"nn&n,h )0 Ph.D.0 S"*er'&s&n, Pro%essor

-ere)& h Her#&h$0 Ph.D.

-ohn -ohnson0 Ph.D.

X&n6Y"n L"0 Ph.D.

G#enn 4. Tone$0 Ph.D.

D n&e# Lod,e0 Ph.D.

He#)"t Gott#e&70 Ph.D.0 E(tern # Ad'&s&n, Pro%essor

D te

APPROVED5

888888888888888888888888888 D '&d /e&ss0 PhD De n0 GSBS


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DEDICATION

To Lo"&se

A 9o) n o% ,re t !o"r ,e nd s"7#&)e d&,n&t$


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ACKNO/LEDGE4ENTS

I 9o"#d #&:e to th n: )$ )entor0 Dr. -. Tho) s C"nn&n,h )0 %or h&s ,"&d n

nd s"**ort d"r&n, the * st %o"r $e rs. I ) #so 'er$ th n:%"# %or the ,"&d n

en!o"r ,e)ent o% )$ 'er$ ! * 7#e !o))&ttee )e)7ers.

I #so 9 nt to th n: !"rrent nd *re'&o"s )e)7ers o% )$ # 7 %or the&

!ontr&7"t&ons5 F# '& C rreno0 D '&d Kn&,ht0 T. Pr sh nt Ned"n, d&0 -oe# L&t

-&. And %&n ##$0 I 9o"#d #&:e to !:no9#ed,e the s"**ort o% the N t&on # InHe #th %or the&r %"nd&n, thro",h ,r nts NIH R31 HL3; 3 DK3?>??@.

5


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DISSERTATION ABSTRACT

SUBFORNICAL ORGAN ANGIOTENSIN TYPE 1 RECEPTOR EXPRESSION

AND DRINKING BEHAVIOR IN BILE DUCT LIGATED RATS

P"7#&! t&on No. 888888888888888888888888

-ose*h D. / #!h0 Ph.D.

The Un&'ers&t$ o% Te( s He #th S!&en!e Center t S n Anton&o

Gr d" te S!hoo# o% B&o)ed&! # S!&en!es

S"*er'&s&n, Pro%essor5 -. Tho) s C"nn&n,h )0 Ph.D.

H$*on tre)& &s the )ost !o))on !#&n&! ##$ s&,n&%&! nt e#e!tro#$te d

D"t&on # h$*on tre)& &s ! "sed 7$ 9 ter retent&on t$*&! ##$0 9h&!h re

&n **ro*r& te ' so*ress&n re#e se &n d&se ses #&:e he rt % "re or #&'er %

L&'er % "re &nd"!es s$ste)&! rter& # ' sod t&on nd ,ener #& ed ede) . 6


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' sod t&on &s h$*othes& ed to ! "se e#e' ted *# s) ren&n !t&'&t$0 ' so*res

nd th&rst. A#tho",h the !entr # )e!h n&s)s th t )ed& te dr&n:&n, 7eh '&or

&n **ro*r& te ' so*ress&n re#e se re not !#e r0 7oth h 'e 7een sho9n to 7e %%

n,&otens&n s&,n #&n, t %ore7r &n s&tes. T9o %ore7r &n !&r!")'entr&!"# r or,

the s"7%orn&! # or, n SFO nd the or, n") ' s!"#os") o% the # )&n ter)&n

OVLT re #o! ted #on, the nter&or 9 ## o% the th&rd !ere7r # 'entr&!#e %"

sensor$ s$ste) th t )on&tors !h n,es &n !&r!"# t&n, hor)ones s"!h s n,&otens&n0

re &)*#&! ted &n ! rd&o' s!"# r nd 7od$ %#"&d ho)eost s&s. Re!ent st"d&es &

the )e!h n&s)s o% dr&n:&n, 7eh '&or nd ' so*ress&n re#e se h 'e sho9n th t

%ore7r &n CVOs re &n'o#'ed &n os)ot&! nd non6os)ot&! ne"r # s&,n #&n, *

Th"s !h n,es &n dr&n:&n, 7eh '&or nd !&r!"# t&n, ' so*ress&n sso!& ted 9

!&rrhos&s ) $ &n'o#'e n,&otens&n II !t&n, t the SFO nd OVLT !ontr&7"t&n

* tho*h$s&o#o,$ o% 9 ter 7 # n!e.

Chron&! 7e d"!t #&, t&on BDL &n the r t &s !o))on#$ "sed )ode# o%

!&rrhos&s th t &s sso!& ted 9&th e#e' ted !&r!"# t&n, ' so*ress&n nd &n!re

ren&n !t&'&t$. As &n the h") n !ond&t&on0 r ts 9&th BDL h 'e e#e' ted !&r

' so*ress&n des*&te 7e&n, h$*er'o#e)&! nd h$*o6os)ot&!. The ,o # o% these st"d&

to deter)&ne )e!h n&s)s &n'o#'ed &n #tered dr&n:&n, 7eh '&or nd ' so*ress&n r

th t #e d to d&sr"*ted h$dro)&ner # 7 # n!e &n the BDL )ode#. 4$ h$*othes&s &s t

n,&otens&n s&,n #&n, d"r&n, BDL !t&' tes do9nstre ) !entr # ne"r # * th9

&n!re se dr&n:&n, 7eh '&or nd ! "se &n **ro*r& te ' so*ress&n re#e se. /e o7


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&n!re sed n,&otens&n II t$*e 1 re!e*tor AT1R e(*ress&on &n the SFO o% B

B sed on th&s o7ser' t&on 9e "sed *h r) !o#o,&! # 7#o!: de o% AT1R nd

sso!& ted '&r # shRNA :no!:do9n o% AT1R e(*ress&on to de)onstr te th t S

n,&otens&n s&,n #&n, 9 s ne!ess r$ %or &n!re sed dr&n:&n, 7eh '&or d"r

!&rrhos&s. Ho9e'er0 !t&' t&on o% ) ,no!e##"# r ne"rose!retor$ !e##s nd ' so*

re#e se 9 s not d&)&n&shed 7$ &nter%er&n, 9&th SFO n,&otens&n s&,n #

o7ser' t&ons s",,est th t n,&otens&n s&,n #&n, thro",h the SFO &s ne"r # )e!h

)ed& t&n, dr&n:&n, 7eh '&or &n n n&) # )ode# o% !on,est&'e #&'er % "re0

o"r "nderst nd&n, o% the * tho*h$s&o#o,$ o% d"t&on # h$*on tre)& .

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TABLE OF CONTENTS

Ded&! t&on...........................................................................................................................

A!:no9#ed,e)ents ..............................................................................................................

D&ssert t&on A7str !t............................................................................................................

T 7#e o% Contents.................................................................................................................

L&st o% t 7#es.......................................................................................................................

L&st o% %&,"res.....................................................................................................................

Ch *ter 15 Gener # Introd"!t&on........................................................................................

Literature Review.........................................................................................................

Bile Duct Ligation Animal Model.........................................................................

Osmotic and Non-osmotic Regulation of Water Intake.........................................

Osmotic and Non-osmotic Regulation of Vasopressin Release............................

Summary................................................................................................................

Ch *ter 5 Intr !ere7ro'entr&!"# r ICV Los rt n &n%"s&on nor) #& es An,&otet$*e 1 re!e*tor AT1R e(*ress&on &n the s"7%orn&! # or, n SFO nd dr&n:& 7eh '&or &n 7e d"!t #&, ted r ts..................................................................................

Abstract........................................................................................................................

Introduction..................................................................................................................

Materials and Methods.................................................................................................

Animals..................................................................................................................

Bile Duct ligation model of hepatic cirrhosis........................................................

Chronic ICV cannula and osmotic minipump implantation..................................

Metabolic cages.....................................................................................................

Plasma measurements............................................................................................

Micropunch dissection of forebrain tissues...........................................................9


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Western Blot..........................................................................................................

RNA extraction and reverse transcription..............................................................

Quantitative real-time PCR....................................................................................

Quantification and analysis of PCR results...........................................................

Experimental protocol............................................................................................

Statistical Analysis.................................................................................................

Results..........................................................................................................................

Effects of BDL on fluid balance and AT1 receptors.............................................

Effects of chronic losartan infusions on fluid balance and AT1 receptor

expression in BDL rats.....................................................................................

Discussion....................................................................................................................

PERSPECTIVES...................................................................................................

Ch *ter >5 An,&otens&n II re!e*tor s"7t$*e 1 AT1 R ,ene :no!:do9n &n ne"ronso% the s"7%orn&! # or, n SFO *re'ents &n!re sed dr&n:&n, 7eh '&or &n 7

#&, ted r ts....................................................................................................................

Abstract........................................................................................................................

Introduction..................................................................................................................

Materials and Methods.................................................................................................

Animals..................................................................................................................

Bile duct ligation model of hepatic cirrhosis.........................................................

Virally mediated AT1a receptor mRNA interference............................................

Metabolic cages.....................................................................................................

Plasma measurements............................................................................................

FosB immunohistochemistry...............................................................................

Confocal imaging and quantification of immunohistochemistry...........................

Laser capture microdissection................................................................................

Quantitative real-time PCR....................................................................................

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Quantification and analysis of PCR results...........................................................

Experimental protocol............................................................................................

Statistical analysis..................................................................................................

Results..........................................................................................................................

Characterization of BDL experimental model and plasma measurements............

AT1aR shRNA viral transduction and gene knockdown.......................................

Fluid intake and renal sodium excretion................................................................

FosB in the SON of BDL rats..............................................................................

Discussion....................................................................................................................

Perspectives.......................................................................................................... 107

Ch *ter @5 S")) r$ nd S&,n&%&! n!e............................................................................ 109

Per*e!t&'es....................................................................................................................... 113

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LIST OF TABLES

Number Page

Table 1: Measurements of serum osmolality, hematocrit, protein, liver weight to body

weight ratio, and final body weight from sham (Sham) and bile duct

ligated (BDL) rats............................................................................................

Table 2: Measurements of serum osmolality, hematocrit, protein, liver weight to body

weight ratio and final body weight from sham (Sham) and bile duct ligated

(BDL) rats that were treated with either vehicle ICV (Veh), losartan ICV(Los), or losartan subcutaneously (SCLos)......................................................

Table 3: RT-qPCR primer sequences................................................................................

Table 4: Measurements of serum osmolality, hematocrit, protein, and liver weight to

body weight ratio in sham (Sham) and bile duct ligated (BDL) rats

injected with a viral construct containing either a scrambled control

shRNA (SCM) or a shRNA targeted to AT1aR (sh-AT1a).............................

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LIST OF FIGURES

Number Page

Figure 1: Diagram showing primary (black arrows) and alternate (light blue arrows)

neural and humoral pathways proposed to mediate increased water intake

and vasopressin release during BDL. The initiating factor is the peripheral

vasodilation that occurs as a result of portal hypertension (see Literature

Review). Peripheral vasodilation eventually decompensates and outstrips

renal autoregulatory ability by decreasing blood flow and resulting in therelease of plasma renin (black arrows). Acting on the SFO, angiotensin II

increases the excitatory drive for increased drinking behavior and

vasopressin release. Light blue arrows show a possible

alternative/redundant pathway involved in the stimulation of AVP release.

Red lettering indicates peripheral factors, and black lettering indicates

central factors. Abbreviations: AT1R, Angiotensin type 1 receptor; SFO,

subfornical organ; OVLT, organum vasculosum of the lamina terminalis;

MnPO, median preoptic nucleus; SON, supraoptic nucleus; NTS, nucleus

of the solitary tract; CVL, caudal ventrolateral medulla..................................

Figure 2: Average daily water intake and urine output (A) and average daily sodium

intake and urinary sodium excretion (B) from Sham and BDL rats. Values

are normalized to body weight (Kg) and represented as means SEM.

*Statistically significant, P


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mean SEM relative protein and mRNA abundance. * Statistically

significant, P


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Figure 9: Representative digital images of the SFO expressing green fluorescent

protein from a rat injected with the viral transduction construct containing

either AAV-GFP-SCM shRNA (A) or AAV-GFP-AT1a shRNA (B) into

the SFO. Scale bar is 50 m.............................................................................

Figure 10: RT-qPCR analysis of AT1aR mRNA from a laser microdissected SFO

nucleus from Sham and BDL rats injected with either a scrambled AAV-

shRNA (SCM) or an AAV-shRNA targeted to AT1a gene (sh-AT1a) into

the SFO. The data are presented as mean SEM relative mRNA levels as

calculated by the 2 -CT method. *Statistically significant, p


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way repeated measures ANOVA with post-hoc interaction test (SNK).

Animal numbers are 12-18 for each group......................................................

Figure 14: Effects of bile duct ligation and AAV-GFP-AT1a shRNA injection into the

SFO on the numbers of cells stained for FosB, vasopressin (AVP) and

colocalized FosB and vasopressin in the supraoptic nucleus of rats in the

several treatment groups. *Significantly different from sham groups

p


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Figure 18: Effects of bile duct ligation and AAV-GFP-AT1a-shRNA injection into

the SFO on the numbers of cells stained for FosB, oxytocin (OXY) and

colocalized FosB and oxytocin in the supraoptic nucleus of rats in the

several treatment groups. *Significantly different from sham groups

p


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vasopressin release due to decompensated peripheral vasodilation (blue

lines) that results from liver cirrhosis (see Ch. 1). Abbreviation: CVL,

caudal ventrolateral medulla; DBH, dopamine- -hydroxylase; MnPO,

median preoptic nucleus; NTS, nucleus of the solitary tract; OVLT,

organum vasculosum of the lamina terminalis; P. Pit., posterior pituitary;

SFO, subfornical organ; SON, supraoptic nucleus........................................ 118

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CHAPTER 15 GENERAL INTRODUCTION

Re,"# t&on o% *# s) os)o# #&t$ nor) ##$ de*ends on t9o *r&n!&*#e )e!h

1 9 ter &nt :e16>0 nd so#"te6%ree 9 ter e(!ret&on )ed& ted 7$ r,&n&ne ' so*

AVP . AVP &s s$nthes& ed &n h$*oth # )&! n"!#e&0 9h&!h then tr ns*orts the *

(on %or re#e se %ro) the *oster&or *&t"&t r$ &nto 7#ood !&r!"# t&on. The ne"ro

s$ste) %or the s$nthes&s nd re#e se o% AVP &nto !&r!"# t&on &s !

ne"roh$*o*h$se # s$ste). C&r!"# t&n, AVP !ts on V re!e*tors o% the :&dne$; to ! "se

+" *or&n !h nne# &nsert&on &nto the *&! # !o##e!t&n, d"!t )e)7r ne ##o

re 7sor*t&on o% 9 ter. In he #th$ h") ns0 *# s) os)o# #&t$ &s re,"# ted 9&

n rro9 r n,e o% ?36 < )Os) :, HO. Under nor) # !ond&t&ons the os)ot&! thresho#d

nd sens&t&'&t$ %or AVP re#e se re s&) r %or th&rst610 nd % !tors th t st&)"# te AVP

re#e se #so ,ener ##$ st&)"# te th&rst. H$*on tre)& %ro) 9 ter &nt :e

"n!o))on "nder nor) # !ond&t&ons &n he #th$ h") ns. Ho9e'er0 there &s e'&den!e th

dr&n:&n, 7eh '&or ) $ *# $ ro#e &n re,"# t&n, 9ho#e 7od$ 9 ter 7 # n!e d"r&n,

d&se se.

H$*on tre)& &s the )ost %re+"ent#$ o!!"rr&n, !#&n&! # e#e!tro#$te d&sor

nn" # tre t)ent !osts &n the US est&) ted to r n,e %ro) 1.; to >.; 7#&3.

H$*on tre)& &s #so sso!& ted 9&th ne, t&'e o"t!o)es &n !hron&! d&se se st

not 7#$ !on,est&'e he rt % "re nd #&'er !&rrhos&s. In 7oth !on,est&'e he rt %

!&rrhos&s0 !h n,es &n the os)ore,"# t&on o% AVP re#e se %ro) the ne"roh$*o*19


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s$ste) !ontr&7"tes to 9 ter retent&on nd d"t&on # h$*on tre)& 66#e d&n, to

)or7&d&t$ nd )ort #&t$36 ?. A#tho",h tre t)ent 9&th s*e!&%&! V re!e*tor nt ,on&

V A &n!re ses so#"te6%ree 9 ter e(!ret&on nd r &ses *# s) os)o# #&t$ &n

% "re nd !&rrhos&s6>10 e'&den!e %or 7ene%&!& # red"!t&on &n !#&n&! # * r )ete

)ort #&t$ or hos*&t #& t&on &s "n!#e r > 6>@.

De!re sed s$ste)&! 7#ood *ress"re h s ) n$ e%%e!ts th t &)* !t 7od$ %

ho)eost s&s. These &n!#"de red"!&n, !t&'&t$ o% #o9 *ress"re 'o#")e re!e*tor %%

&n!re s&n, *# s) ren&n re#e se0 nd &n!re s&n, s$)* thet&! ner'e d&s!h

ne"ro,en&! ' so)otor tone. Th&s ne"roh")or # !t&' t&on *ro%e &s #so %

n&) # )ode#s o% !&rrhos&s>;6@. While this explanation is generally accepted in the

literature, the precise neural and molecular factors that affect the downward resetting

of the drinking behavior and AVP osmotic thresholds remain undetermined. I)*ro'ed

"nderst nd&n, o% d$s%"n!t&on # os)ore,"# t&on o% th&rst nd AVP re#e se &)&,ht &)*ro'e d&se se * ##& t&on0 tre t)ent re,&)ens th t )&n&)& e d'erse dr", e

nd &)*ro'e !#&n&! # o"t!o)es.


!&rrhos&s. As &n the h") n !ond&t&on0 these r ts h 'e e#e' ted !&r!"# t&n, ' s

des*&te &n!re sed *os&t&'e 9 ter 7 # n!e0 h$*er'o#e)& 9&th s!&tes %or)h$*on tre)& . 4$ d t re !ons&stent 9&th *re'&o"s st"d&es de)onstr t&n, th

&n!re sed #e'e# o% *# s) AVP &s d"e to enh n!ed !t&'&t$ o% the ne"roh$*o*

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s$ste). The purpose of this pro ect is to more fully elucidate neural mechanisms that

influence regulation of spontaneous water intake and vasopressin release in the !"#

rat model of cirrhosis.

LITERATURE REVIEW

BILE DUCT L IGATION ANIMAL M ODEL

The *ort # !&r!"# tor$ s$ste) &s one o% t9o "n&+"e !&r!"# tor$ s$ste)s &

) )) # 9here t9o ! *# r$ 7eds re !onne!ted &n ser&es. The *ort # 'eno"s s$st

re!e&'es s*# n!hn&! 7#ood %#o9 %ro) the s"7*hren&! se,)ent o% the d&,est&'e tr !:

to the re!t")0 the * n!re s nd the s*#een. D"r&n, #&'er !&rrhos&s0 *ort # h$*ert

res"#ts %ro) 7oth &n!re sed &ntr he* t&! res&st n!e to 7#ood o"t%#o9 nd &

s*# n!hn&! &n%#o9@. L&'er !&rrhos&s &n!re ses &ntr he* t&! res&st n!e t

&n%# )) tor$ !t&' t&on o% he* t&! ste## te !e##s 9h&!h enh n!es the&r !ontr

&n!re ses s&n"so&d # res&st n!e. I)* &red 7#ood %#o9 thro",h the #&'er res"#ts &

9h&!h &nd"!es !o)*ens tor$ res*onses &n the he* t&! rter$ to &n!re se 7#ood %#o

&n!re sed 7#ood %#o9 &s % !&t ted 7$ d&%%"s&on o% denos&ne to ' sod&

rter&o#es 7$ &n!re s&n, the *rod"!t&on o% n&tr&! o(&de NO . Prod"!t&on o%

&n the s&n"so&d # nd *osts&n"so&d # re s #e d&n, to &n!re sed &ntr he* t&

the se+"ester&n, o% 7#ood 9&th&n the #&'er


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A**ro(&) te#$ < o% ! rd& ! o"t*"t CO &s re!e&'ed 7$ s*# n!n&! !&r!"# t&

There%ore0 *ort # h$*ertens&on res"#ts %ro) 7oth &n!re sed &ntr he* t&! res&

he* t&! rter$ ' sod t&on. In %"#)&n te !&rrhos&s0 *ort # !&r!"# t&on re'erse

&n!re sed rter&o'eno"s nd 'eno'eno"s sh"nt&n, thro",h !o## ter # !&r!"#

enh n!&n, s*# n!n&! 7#ood %#o9;>.

The de'e#o*)ent o% *ort # h$*ertens&on #e ds to s*# n!n&! h$*ere)&

)e!h n&! # stress on endothe#& # !e##s st&)"# t&n, denos&ne nd NO *rod"!;@6;;.

Chron&! ##$ e#e' ted *ort # *ress"re nd s*# n!n&! ' sod t&on #on, 9&th the

o% !o## ter # !&r!"# t&on &nd"!es st te o% h$*erd$n )&! !&r!"# t&on !h r

&n!re sed s$ste)&! %#o9 nd *er&*her # ' sod t&on )ed& ted 7$ NO. H$*e

!&r!"# t&on %%e!ts other or, n s$ste)s &n the 7od$ nd &s !h r !ter& ed 7$ &n!r

r te0 ! rd& ! o"t*"t0 *# s) 'o#")e0 ren # sod&") retent&on0 nd *ro,ress&'e *er&

' sod t&on. It h s 7een s",,ested th t s*# n!hn&! nd s$ste)&! ' sod t&on th tthe h$*erd$n )&! !&r!"# tor$ st te or&,&n tes s se!ond r$ ne"r # *heno)enon

he* t&! 'o#")e *ress"reJ %%erents. Ho9e'er0 there **e rs to 7e )ore e'&den!e %o

*rod"!t&on o% ' sod tor$ % !tors s"!h s denos&ne nd NO thro",h she

st&)"# t&on o% endothe#& # !e##s s res"#t o% &n!re sed s*# n!hn&! nd s$

%#o9 s n e(*# n t&on %or the *er&*her # ' sod t&on0 sod&") ret

h$*erd$n )&! !&r!"# t&on seen &n !&rrhos&s2162@.

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The 'eno"s s$ste) *# $s n &)*ort nt ro#e &n s"**ort&n, 7#ood *ress"re

) &nt &n&n, reser'e o% 7#ood 'o#")e th t ! n 7e )o7& ed d"r&n, t&)es o% de

*er%"s&on. S*# n!n&! nd !"t neo"s 'e&ns re the )ost !o)*#& nt nd re*resent the

7#ood 'o#")e reser'o&rs &n the h") n 7od$. D"r&n, !&rrhos&s there &s n &n!re se %

7#ood &nto the h&,h#$ !o)*#& nt s*# n!n&! 'eno"s s$ste) !o"*#ed 9&th n &n!

&ntr he* t&! res&st n!e to o"t%#o9. The !o)7&ned e%%e!t o% &n!re sed s*# n!n

de!re sed he* t&! !ond"!t n!e !!o)* n&ed 7$ !hron&! *er&*her # ' sod t&on &

h$*o'o#e)& nd #&)&ted ! rd& ! *re#o d reser'e.

Pro,ress&'e de!o)*ens t&on o% *er&*her # ' sod t&on &n !&rrhos&s #e

sh&%t&n, to e(tr ' s!"# r e.,.0 s!&tes or 'eno"s !o)* rt)ents thro",h &n!re sed 'en

! * !&t n!e nd !entr # h$*o'o#e)& . E(tr !e##"# r 'o#")e &s deter)&ned 7$

sod&") h nd#&n,0 9h&!h re,"# tes the tot # 7od$ sod&") !ontent. S!hr&er *ro*ose

"n&%$&n, h$*othes&s o% e(tr !e##"# r 'o#")e re,"# t&on &n he #th nd d&se!on!e*t :no9n s e%%e!t&'e rter& # 7#ood 'o#")e EABV . The # tter re*resents %

o% the rter& # ' s!"# r !o)* rt)ent nd &s ) or deter)&n nt o% ren # re,"#

sod&") nd 9 ter e(!ret&on0 9h&!h )od"# tes e(tr !e##"# r %#"&d 'o#")e nd os)o#

A!!ord&n, to S!hr&erJs "n&%$&n, h$*othes&s0 *ro,ress&'e *er&*her # ' so

e'ent" ##$ o"tstr&*s the ! * !&t$ %or ! rd& ! !o)*ens t&on nd res"#ts &n de!re

7#ood %#o90 &n!re sed %tr t&on %r !t&on0 nd de!o)*ens t&on o%

)e!h n&s)s &n !ond&t&on :no9n s he* toren # s$ndro)e. In!re sed ren # so

retent&on nd ren # d$s%"n!t&on ! n 7e !orre!ted 7$ ' so*ressors th t !t ) &n23


24/144

&n!re se *er&*her # ' s!"# r res&st n!e nd &n!re se ren # *er%"s&on *ress?@. Th&s

s"**orts the h$*othes&s th t the et&o#o,$ o% ren # sod&") retent&on &s *e

' sod t&on?@. Ne"r # %%erents %ro) "n#o ded *ress"re 'o#")e re!e*tors st&)"#

ne"roh")or # res*onses &n!#"d&n, !t&' t&on o% the s$)* thet&! ner'o"s s$s

&n!re sed *# s) ren&n !t&'&t$ nd non6os)ot&! AVP re#e se. Th&s *ro

ne"roh")or # !t&' t&on ! n #e d to d"t&on # h$*on tre)& 0 9h&!h !o

s&,n&%&! nt#$ to de7&t t&n, )or7&d&t$ nd )ort #&t$ o% !&rrhos&s. Re,"#

AVP &s 'er$ sens&t&'e to s) ## !h n,es &n *# s) os)o# #&t$0 7"t re+"&res

red"!t&ons o% 7#ood 'o#")e nd or *ress"re to st&)"# te e+"&' #ent re#e se1. As !&rrhos&s

*ro,resses0 *er&*her # ' sod t&on *rod"!es )ore *rono"n!ed ne"roh")or # !t&'

F"rther)ore0 s$ste)&! h$*otens&on h s s&) r re,"# tor$ e%%e!t on 9 ter

Hen!e0 there &s e'&den!e th t s se'er&t$ o% d&se se *ro,resses0 the *r&) r$ #o

tre t)ent %or d"t&on # h$*on tre)& e.,.0 9 ter restr&!t&on 7e!o)es #ess to#e

e%%e!t&'e6 @. 4e!h n&s)s o% &)* &red %ree 9 ter !#e r n!e &n !&rrhos&s &n!#"

)&nor % !tors s"!h s de!re sed ,#o)er"# r %tr t&on nd &n!re sed sod&") re

&n the *ro(&) # t"7"#e


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O SMOTIC AND NON -OSMOTIC R EGULATION OF W ATER I NTAKE

In he #th$ n&) #s0 the !entr # ne"r # )e!h n&s)s th t re,"# te 7#ood 'o#")e

os)o# #&t$ re &nter!onne!ted. The s*e!&%&!&t$ o% o'er# * 7et9een the %

e%%erent ho)eost t&! res*onses &s re'e #ed 7$ *h$s&o#o,&! # !h ##en,es. For

ne"r # * th9 $s th t res*ond to # r,e de!re ses &n 7#ood *ress"re or 'o#")e ! n &n!r

9 ter &nt :e or ' so*ress&n re#e se0 9h&!h then de!re ses *# s) os)o#

A!!ord&n,#$0 d&se ses th t res"#t &n !hron&! ##$ de!re sed 7#ood *ress"re re

9&th non6os)ot&! !t&' t&on o% * th9 $s th t re,"# te h")or # os)ot&! ho)eos

there7$ de!re s&n, *# s) os)o# #&t$.

The # )&n ter)&n #&s0 re,&on o% the 7r &n s&t" ted &n the )&d#&ne

o% the th&rd 'entr&!#e0 !ont &ns t9o !&r!")'entr&!"# r or, ns5 the or, n") ' s!"#os")

the # )&n ter)&n #&s OVLT nd the s"7%orn&! # or, n SFO . Ne"rons 9&

"n&+"e %ore7r &n re,&ons re s"rro"nded 7$ %enestr ted ! *# r&es th t # !:

7#ood 7r &n 7 rr&er 131613>0 9h&!h ##o9s these str"!t"res to 7e sens&t&'e to !&r!"# t&

% !tors s"!h s *# s) os)o# #&t$ nd n,&otens&n13@6132. The OVLT nd SFO 7oth *ro e!t

to ' r&o"s h$*oth # )&! s&tes nd dense#$ to the )ed& n *reo*t&! n"!#e"s 4nPO

&s n &)*ort nt &nte,r t&'e !enter %or h$dro)&ner # nd ! rd&o' s!"# r re,

E'&den!e &n ,o ts13?0 do,s0 rodents111611>0 nd shee*11@611; s",,ests th t the OVLT *# $s

*redo)&n nt ro#e &n the tr nsd"!t&on o% os)ot&! s&,n #s thro",h ne!ess r$ * th9

&n!#"des the 4nPO. Ho9e'er0 &n shee* se#e!t&'e #es&on o% the SFO0 OVLT

!o)7&ned 7# t&on o% 7oth d&d not 7o#&sh the d&*so,en&! res*onse to n25


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st&)"#"s11;. Interest&n,#$0 !o)*#ete 7# t&on o% SFO0 OVLT nd 4nPO d&d

h$*eros)ot&! st&)"# ted dr&n:&n,. /here s 7# t&on o% OVLT nd 4nPO &n!#"

7# t&on o% the ent&re ntero'entr # 9 ## o% the th&rd 'entr&!#e h s res"#ted &n

d&*s& 0 7 se#&nead libitum 9 ter &nt :e nd !&r! d& n rh$th)s &n dr&n:&n, 7eh '&or

not *er) nent#$ #tered11;. St"d&es th t &n!#"ded &))"nost &n&n, o%c-fos0 *roto6

on!o,ene ) r:er o% ne"ron # !t&' t&on0 #so !ontr&7"te to e'&den!e th t SFO nd

ne"rons re !t&' ted 7$ h$*erton&!&t$ nd th t the 4nPO &s !t&' ted se!ond r

SFO nd OVLT !t&' t&on11261 3.

An,&otens&n h s #on, 7een :no9n %or &ts d&*so,en&! *ro*ert&es0 nd the &

o% 7oth SFO nd 4nPO ne"rons re essent& # %or *er&*her # n,&otens&n6&nd

&nt :e. Ne"ro n to)&! # st"d&es "s&n,c-fos h 'e sho9n th t SFO ne"ron !t&' t&on 7$

7oth &ntr 'eno"s n,&otens&n II1 ?61>1 nd &so*rotereno# 9h&!h st&)"# tes ren&n se!ret

d&re!t#$ nd &nd&re!t#$ 7$ #o9er&n, rter& # 7#ood *ress"re 0 nd th t !t&' t&sso!& ted 9&th &n!re sed 9 ter &nt :e. There &s #so e'&den!e th t the ne"rons #

the o"ter 7order o% the SFO0 9h&!h re !t&' ted 7$ d&*so,en&! doses o% n,&ot

&so*rotereno#0 *ro e!t to 4nPO ne"rons1>@61>;.

Intr !ere7ro'entr&!"# r ICV d)&n&str t&on o% n,&otens&n II #so

st&)"# tes dr&n:&n, &n r ts0 nd the 4nPO **e rs to 7e ne!ess r$ %or the d&*e%%e!t o% !entr # n,&otens&n II1>261>. Other st"d&es "t& &n, tr ns,en&! )&!e h 'e sho

th t the &n!re sed *rod"!t&on o% SFO n,&otens&n II &n!re sed 9 ter &nt :e1@30 nd th t

26


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!hron&! 7r &n6restr&!ted o'ere(*ress&on o% AT1R res"#ts &n h&,her 7 se#&ne 9

nd n &n!re sed 9 ter &nt :e res*onse to e(o,eno"s ICV n,&otens&n II1@1. St"d&es o%

s*ont neo"s#$ h$*ertens&'e r ts1@ 61@@0 r ts de*r&'ed o% 9 ter 1@


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!t&'&t$ o% ' so*ress&n ne"rons &n the SON thro",h the # )&n ter)&n #&s. Ho9

'&'o st"d&es s",,est th t %%erent *ress"re 'o#")e &n*"ts %ro) the )ed"## see) to *

n &)*ort nt st&)"# tor$ ro#e d"r&n, !"te h$*otens&on nd he)orrh ,e to &n!

AVP1< 61;;. There%ore0 AVP re#e se d"r&n, !"te nd !hron&! 7o"ts o% h$*otens&on

res"#ts %ro) )"#t&*#e red"nd nt ho)eost t&! )e!h n&s)s0 nd ) $ 7e )ore !o)*#e( th n

s&n,#e ho)eost t&! re%#e( #oo*. Th&s ) :es sense !ons&der&n, the '&t # &)*or

!ont&n"o"s or, n *er%"s&on %or s"r'&' #.

In *re'&o"s e(*er&)ent *er%or)ed &n o"r # 70 ,ro"* o% BDL r ts 9ere ,&

s #&ne to dr&n: to &n!re se s #t de#&'er$ to the d&st # t"7"#e th"s de!re s&

ren&n !t&'&t$ PRA thro",h the ) !"# dens 6 "(t ,#o)er"# r ** r t"s )e!h n&1;2.

H&,h s #t &nt :e 9 s !!o)* n&ed 7$ s&,n&%&! nt de!re se &n !&r!"# t&n,

9 s sso!& ted 9&th de!re se &n PRA !o)* red to BDL r ts th t dr n: on#$ 9 t1;2.

E r#&er st"d&es sho9ed th t ne"rons nd ,#& o% the # )&n ter)&n #&s to,ether%"n!t&on # ren&n6 n,&otens&n s$ste) th t ) $ *er)&t n,&otens&n II re#e se &n

these ne"r # *ro e!t&ons1;?61210 nd &n '&tro e#e!tro*h$s&o#o,&! # st"d&es h 'e #so s

th t n,&otens&n ! n !t *ost6s$n *t&! ##$ to !t&' te non6s*e!&%&! ! t&on&! !

de*o# r& e SON ne"rons nd *re6s$n *t&! ##$ to % !&t te ,#"t ) te re#e s123612. Th&s

s",,ests th t !&r!"# t&n, n,&otens&n )&,ht !ontr&7"te to &n **ro*r& te re#e se o%

!t&n, on ne"rons o% %ore7r &n !&r!")'entr&!"# r or, ns0 ho9e'er th&s h$*othes&s

7een tested. To !# r&%$ the ro#e o% n,&otens&n &n the st&)"# t&n, AVP re#e s

&nt :e I h 'e %o!"sed on !t&ons o% !&r!"# t&n, n,&otens&n &n SFO.28


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4$ 9or:&n, h$*othes&s &s th t !&r!"# t&n, n,&otens&n !t&n, on ne"rons o

&nd"!es ne"r # d *t t&ons e.,.0 "*re,"# t&on o% AT1R e(*ress&on th t

&n!re sed dr&n:&n, 7eh '&or sso!& ted 9&th BDL F&,"re 1 . The ro#e o%

re!e*tors &n )ed& t&n, AVP re#e se nd 9 ter retent&on sso!& ted 9&th BDL 9 s

r ts 7$ &n e!ted o% AT1R shRNA nd &n%"s&on o% #os rt n &nto the !ere7r # 'en

SUMMARY

G &n&n, ,re ter "nderst nd&n, o% ho9 9 ter &nt :e nd ' so*ress&n re#e

re,"# ted os)ot&! ##$ nd non6os)ot&! ##$ !o"#d he#* to &)*ro'e tre t)ent *roto!o#

* t&ents 9&th #&'er !&rrhos&s0 *otent& ##$ *re'ent&n, or re'erse&n, d"t&on # h

F"rther e#"!&d t&on o% the )e!h n&s)s th t re,"# te 9 ter &nt :e 9# #so &n%

9 $s to *re'ent d'erse dr", e%%e!ts s 9e## s * ##& te e%%e!ts o% d&se se *ro,r

#e d to &n **ro*r& te dr&n:&n, 7eh '&or.

29


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Figure 1: Diagram showing primary (black arrows) and alternate (light blue arrows)

neural and humoral pathways proposed to mediate increased water intake and vasopressinrelease during BDL. The initiating factor is the peripheral vasodilation that occurs as aresult of portal hypertension (see Literature Review). Peripheral vasodilation eventuallydecompensates and outstrips renal autoregulatory ability by decreasing blood flow andresulting in the release of plasma renin (black arrows). Acting on the SFO, angiotensin IIincreases the excitatory drive for increased drinking behavior and vasopressin release.Light blue arrows show a possible alternative/redundant pathway involved in thestimulation of AVP release. Red lettering indicates peripheral factors, and black letteringindicates central factors. Abbreviations: AT1R, Angiotensin type 1 receptor; SFO,subfornical organ; OVLT, organum vasculosum of the lamina terminalis; MnPO, medianpreoptic nucleus; SON, supraoptic nucleus; NTS, nucleus of the solitary tract; CVL,caudal ventrolateral medulla.

30


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CHAPTER 5 INTRACEREBROVENTRICULAR ICV LOSARTAN INFUSION NOR4ALIMES ANGIOTENSIN TYPE 1 RECEPTOR AT1R EXPRESSION IN

THE SUBFORNICAL ORGAN SFO AND DRINKING BEHAVIOR IN BILEDUCT LIGATED RATS

A77re'& ted t&t#e5 In!re sed dr&n:&n, 7eh '&or nd AT1R d"r&n, &n **ro*r& te ' so*ress&

re#e se

Authors: -ose*h D / #!h10 0 F# '& Re,&n C rre o0 nd -. Tho) s C"nn&n,h )1

1De* rt)ent o% Inte,r t&'e Ph$s&o#o,$0 nd C rd&o' s!"# r Rese r!h Inst&t"teUn&'ers&t$ o% North Te( s He #th S!&en!e Center t Fort /orth0> F#o$d C"r# Dr&'eS n Anton&o0 TX 2?

31


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ABSTRACT

Be d"!t #&, t&on BDL ! "ses !on,est&'e #&'er % "re nd

he)od$n )&! !h n,es &n!#"d&n, *er&*her # ' sod t&on nd ,ener #& ed

Per&*her # ' sod t&on &s h$*othes& ed to then !t&' te !o)*ens tor$ )e!h

&n!#"d&n, &n!re sed %#"&d e#e!tro#$te d$sre,"# t&on nd ne"roh")or # !t&' t&o

&n **ro*r& te r,&n&ne ' so*ress&n AVP re#e se nd &n!re sed *# s) ren

A!t&' t&on o% hor)on # nd ne"r # * th9 $s se!ond r$ to *er&*her # ' sod t&

,ener ##$ tho",ht to &n!re se dr&n:&n, 7eh '&or ho9e'er0 the *re!&se !entr # )e!h

th t ,o'ern ' so*ress&n re#e se nd dr&n:&n, 7eh '&or re not 9e## !h r !ter& ed

!onte(t o% #&'er % "re. The !&r!")'entr&!"# r or, ns0 the S"7%orn&! # Or, n

the or, n") ' s!"#os") o% the # )&n ter)&n #&s OVLT 0 %"n!t&on s sensor$

th t )on&tors !h n,es &n !&r!"# t&n, hor)ones s 9e## s *# s) os)o# #&t$ n

7een &)*#&! ted &n ! rd&o' s!"# r !ontro#0 nd 7od$ %#"&d ho)eost s&s. /e te

h$*othes&s th t &n!re sed !entr # n,&otens&n s&,n #&n, !ontr&7"tes to &n!re s

7eh '&or nd &n **ro*r& te AVP re#e se &n the BDL )ode# o% !&rrhos&s &n t

%&rst "nder9ent BDL or sh ) #&, t&on s"r,er$. A%ter 2 d $s o% re!o'er$0 o

)&n&*")*s %#ed 9&th the n,&otens&n AT1 re!e*tor AT1R nt ,on&st #o

, # or 3. s #&ne 'eh&!#e !onne!ted 7$ t"7&n, to n &ntr !ere7ro'entr&!"#

! nn"# 9ere &)*# nted %or !hron&! &n%"s&on. Se* r te sets o% !ontro#

!hron&! ##$ &n%"sed s"7!"t neo"s#$ 9&th the s )e dose o% #os rt n or 'eh&!#e. St

d $s %ter BDL or sh ) #&, t&on s"r,er$0 r ts 9ere ho"sed &n )et 7o#&! !h )7e32


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)e s"re)ent o% %#"&d nd %ood &nt :e nd "r&ne o"t*"t. On the ?th d $0 the r ts 9ere

nesthet& ed nd de! *&t ted0 nd s )*#es o% 7#ood nd 7r &n *"n!hes 9ere !o#

+" nt&%&! t&on o% AT1R e(*ress&on 7$ 9estern 7#ot nd re #6t&)e +" nt&t t&

+PCR . A'er ,e 7 se#&ne 9 ter &nt :e s&,n&%&! nt#$ &n!re sed &n BDL r ts !

sh )0 nd th&s 9 s tten" ted 7$ !hron&! ICV #os rt n &n%"s&on. U*re,"# t&on

*rote&n nd AT1 R )RNA 9ere o7ser'ed &n the SFO o% BDL r ts !o)* red to sh ).

the OVLT o% BDL n&) #s0 AT1R *rote&n 9 s #so e#e' ted 7"t 9&tho"t n sso

&n!re se &n AT1 R )RNA. Chron&! ICV #os rt n &n%"s&on *re'ented the &n!re se

*rote&n &n the SFO 9&tho"t !h n,&n, *# s) os)o# #&t$. Ren # sod&") retent&

s&,n&%&! nt#$ &n!re sed &n BDL n&) #s0 nd there 9 s no d&%%eren!e &n

&nt :e. D t *resented here &nd&! te *oss&7#e ro#e %or &n!re sed # )&n ter)&

e(*ress&on &n s"**ort&n, &n!re sed dr&n:&n, 7eh '&or d"r&n, !on,est&'e !&rrhos&

s",,est !entr # n,&otens&n s&,n #&n, &s not the *r&) r$ )e!h n&s) res*ons&7

!&rrhos&s6&nd"!ed d"t&on # h$*on tre)& .

33


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INTRODUCTION

P# s) os)o# #&t$ &s nor) ##$ re,"# ted 7$ t9o *r&n!&*#e )e!h n&s)s5 1

&nt :e0 nd so#"te6%ree 9 ter e(!ret&on )ed& ted 7$ r,&n&ne ' so*ress&n

he #th$ h") ns0 *# s) os)o# #&t$ &s ) &nt &ned 9&th&n n rro9 r n,e o%

)Os) :,. Under nor) # *h$s&o#o,&! # !ond&t&ons the os)ot&! thresho#d nd sens&

%or AVP re#e se nd th&rst re s&) r 1 0 nd h$*on tre)& %ro) e(!ess&'e 9 ter &nt :e &

"n!o))on. Ho9e'er0 st"d&es o% the der n,e)ent o% th&rst nd AVP re#e se &nd&! teth&rst &n )7"# tor$ h") ns th t h 'e !!ess to 9 ter &s n &)*ort nt % !tor &

!hron&! re,"# t&on o% 9ho#e 7od$ 9 ter 7 # n!e.

H$*on tre)& &s sso!& ted 9&th ne, t&'e o"t!o)es &n ) n$ !hron&! d&

st tes s"!h s !on,est&'e he rt % "re nd !&rrhos&s. In 7oth ! ses0 &n **ro*r&

AVP re#e se %ro) the *oster&or *&t"&t r$ &n re# t&on to the *re' &n, #o9 #e'e#os)o# #&t$ !ontr&7"tes to 9 ter retent&on nd d"t&on # h$*on tre)&

&n!re s&n, )or7&d&t$ nd )ort #&t$ . Tre t)ent 9&th s*e!&%&! V re!e*tor n

V A &n!re ses so#"te6%ree 9 ter e(!ret&on nd &)*ro'es *# s) os)o# #&t$

% "re nd !&rrhos&s6>10 ho9e'er there h 'e 7een re*orts o% &n!re sed dr&n:&n, 7eh '&

res"#t&n, %ro) d&se se nd *oss&7#$ s s&de6e%%e!t o% V A tre t)ent th t !o

&n!re sed )ort #&t$ nd re6hos*&t #& t&on .

34


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Tr d&t&on ##$0 &n!re sed ne"roh")or # !t&' t&on sso!& ted 9&th !&

7een ttr&7"ted to de!re sed e%%e!t&'e !&r!"# t&n, 'o#")e nd h$*erd$n )&! !&r

s$ndro)e !h r !ter& ed 7$ &n!re sed *ort # h$*ertens&on &n&t& t&n, s*# n!n&

nd !"#)&n t&n, &n *er&*her # ' sod t&on nd ne"roh")or # e(!&t t&on. H$

!&r!"# tor$ s$ndro)e &s re*orted to !t&' te the ren&n6 n,&otens&n s$ste) d"r&n,

!&rrhos&s 9h&!h &s !ons&stent 9&th the tr d&t&on # '&e9 th t non6os)ot&! %

' so*ress&n re#e se sso!& ted 9&th th&s d&se se *oss&7#$ 7$ resett&n, th

thresho#d or , &n. A #&:e#$ ! nd&d te )e!h n&s) &s &n!re sed !&r!"# t&n, n,&

An, II th t !o"#d !ontr&7"te to !h r !ter&st&! s$)* tho6e(!&t t&on0 &n!re

re#e se nd e#e' ted %#"&d &nt :e o% !&rrhos&s 7$ !t&n, on CNS !&r!")'entr&!"#

The # )&n ter)&n #&s !ont &ns t9o !&r!")'entr&!"# r or, ns5 the or,

' s!"#os") o% the # )&n ter)&n #&s OVLT nd the s"7%orn&! # or, n SFO

9&th&n these "n&+"e %ore7r &n re,&ons # !: %"n!t&on # 7#ood 7r &n 7 rr&erne"rons !o)*r&s&n, these str"!t"res to sense nd res*ond to !&r!"# t&n, % !tors s"!

*# s) os)o# #&t$ nd An, II12


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the th&rst nd AVP os)ot&! set6*o&nts do9n9 rd d"r&n, he rt or #&'er % "re

"ndeter)&ned.


!&rrhos&s . As &n the h") n !ond&t&on0 these r ts h 'e e#e' ted !&r!"# t&n, ' s

des*&te s&,n&%&! nt#$ de!re sed *# s) os)o# #&t$0 &n!re sed PRA0 nd h$

s!&tes %or) t&on. Pre'&o"s#$0 o"r # 7 re*orted th t there 9 s s&,n&%&!

re# t&onsh&* 7et9een PRA nd AVP re#e se0 nd th t de!re s&n, PRA 7$ restr&!t

&nt :e to h$*erton&! s #&ne s&,n&%&! nt#$ de!re sed !&r!"# t&n, AVP1;2.

The *"r*ose o% th&s st"d$ 9 s to e' #" te the * ttern o% e(*ress&on o%

)RNA nd *rote&n &n the # )&n ter)&n #&s sso!& ted 9&th BDL &nd"!ed h$*oo

/e %"rther tested the ro#e o% !entr # AT1R !t&'&t$ *h r) !o#o,&! ##$ 9&th

#os rt n &n%"s&ons to %"n!t&on ##$ test the !ontr&7"t&on o% !entr # n,&otens

the de'e#o*)ent o% d"t&on # h$*on tre)& d"r&n, BDL !&rrhos&s.

MATERIALS AND METHODS

ANIMALS

E(*er&)ents 9ere !ond"!ted on d"#t ) #e S*r ,"e6D 9#e$ r ts C en'&ron)ent "nder 1 1 h #&,ht !$!#e 9&

onset t 3233h. E(!e*t d"r&n, s"r,&! # *ro!ed"res0 r ts h dad libitum !!ess to 9 ter nd36


37/144

%ood. The # tter !ont &ned 3.> N C#. A## e(*er&)ent # *ro!ed"res 9ere !ond"!

!!ord n!e 9&th the ,"&de#&nes o% the P"7#&! He #th Ser'&!e nd 9ere **ro'ed

Un&'ers&t$ o% North Te( s He #th S!&en!e Centre Inst&t"t&on # An&) # C re

Co))&ttee.

BILE DUCT LIGATION MODEL OF HEPATIC CIRRHOSIS

E !h n&) # 9 s nesthet& ed 9&th &so%#"r ne &n o($,en . On

nesthet& ed the 7do)en 9 s sh 'ed nd the re 9 s !#e ned 9&th &od&ne nt&s

)&d#&ne &n!&s&on 9 s *er%or)ed on the 7do)en nd the !o))on 7e d"!t 9 s &s

7$ 7#"nt d&sse!t&on nd !"t 7et9een t9o #&, t"res. E !h r t 9 s then ret"rned to

nd )on&tored d $ "nt "sed %or the e(*er&)ent. V&s" # &ns*e!t&on o% s!&t&

*er&tone # ! '&t$ 9 s *er%or)ed d $ %ter s"r,er$. An$ r t sho9&n, )or7&d&

% "re to thr&'e0 s&,n&%&! nt de!re se &n %ood or 9 ter &nt :e 9 s e"th

th&o7"t 7 r7&t # In !t&n 0 S&,) 0 St. Lo"&s0 4O 133 ), :, &.*. . Sh ) #&, te

9ere s"7 e!ted to the s )e s"r,&! # *ro!ed"re 9&th the e(!e*t&on th t the 7e d"!t 9

not #&, ted or !"t. R ts 9ere e"th n& ed @ 9ee:s ? d $s %ter BDL or sh ) s"r,e

!o##e!t&on o% 7r &n nd 7#ood s )*#es. L&'er %&7ros&s 9 s o7ser'ed &n r ts 9&t

7e d"!t #&, t&on. L&'er 7od$ 9e&,ht r t&o nd d&s!o#or t&on o% 7#ood ser")

'er&%$ the de'e#o*)ent o% he* t&! !&rrhos&s.

37


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C HRONIC ICV CANNULA AND OSMOTIC MINIPUMP IMPLANTATION

R ts 9ere ##o9ed to re!o'er %or 2 d $s %ter BDL or sh ) s"r,er$. There %

,ro"*s o% BDL nd sh ) r ts 9ere &nstr")ented 9&th os)ot&! )&n&6*")*s A# et )o

33@0 3. < # hQ1 C"*ert&no0 CA0 USA %or &ntr !ere7ro'entr&!"# r

s"7!"t neo"s SC &n%"s&ons. The # tter 9 s to !ontro# %or *er&*her # dr", e%%

9ere %#ed 9&th e&ther #os rt n , # d&sso#'ed &n 3. s #&ne 'eh&

#one. The dose o% #os rt n 9 s !hosen 7 sed on *re'&o"s &n '&'o st"d&es th t sh

to h 'e no e%%e!t on rter& # *ress"re. P")*s 9ere &n!"7 ted %or t #e st @? h &

9 ter 7 th nd 9e&,hed *r&or to &)*# nt t&on. S"r,er&es 9ere *er%or)ed "s&n,

te!hn&+"e 9&th r ts n esthet& ed 9&th &so%#"r ne. For ICV &n%"s&ons0

!onne!ted 7$ *o#$eth$#ene t"7&n, to *&e!e o% , ",e st &n#ess stee# t"7&n,

One0 Ro no:e0 VA 0 !"t


39/144

M ETABOLIC CAGES

To )e s"re 9 ter &nt :e0 %ood &nt :e0 nd "r&ne o"t*"t0 r ts 9ere ho"se

)et 7o#&! ! ,es L 7 Prod"!ts In!.0 Se %ord0 DE st rt&n, on the 1?th d $ %ter BDL or

sh ) s"r,er$. The )et 7o#&! ! ,e *roto!o# !ons&sted o% 13 d $s. D $s 16> 9

!!#&) t&on d $s &n 9h&!h no )e s"re)ents 9ere t :en. On D $s @6130 %ood &

9 ter &nt :e nd "r&ne o"t*"t 9ere re!orded t 533 ). Food 9 s )e s"red 7$ %#&

%ood !ont &ners to s*e!&%&ed 9e&,ht &n ,r )s nd s"7tr !t&n, the re) &n&n, 9

ho"rs # ter. Sod&") &nt :e 9 s ! #!"# ted %ro) sod&") !ontent o% the %ood 3.>

9e&,ht0 C tW L4@?


40/144

os)o)eter /es!or In!. Lo, n0 UT . Ser") *rote&n 9 s )e s"red 7$ re%r !to)eter$

N t&on # Proto)eter0 N t&on # Instr")ents0 B #t&)ore0 4D .

M ICROPUNCH DISSECTION OF FOREBRAIN TISSUES

T&ss"e !ont &n&n, SFO0 OVLT0 nd 4nPO 9ere )&!ro*"n!hed %ro) the %ore7

%or RT6+PCR nd /estern 7#ot n #$s&s. E !h r t 9 s nesthet& ed In !t&n 1

&.*. nd de! *&t ted. The &so# ted 7r &n 9 s *# !ed &n !o))er!& ##$ ' &

) tr&( Stoe#t&n,0 /ood D #e0 IL . The ) tr&( 9 s "sed to !"t the 7r &n &nto

!oron # s# 7s 9&th r or 7# des. Then0 the des&red re,&ons %ro) the **ro*r&

! "d # 16))6th&!: s#&!e 9ere )&!ro*"n!hed 9&th 16)# s$r&n,es e+"&**ed 9&th 7#"n

, ",e need#es. The *"n!h s )*#es 9ere e(*e##ed &nto )&!ro!entr&%",e t"7es nd sn

%ro en on dr$ &!e.

W ESTERN BLOT

4od&%&ed 9estern 7#ot 9 s *er%or)ed s *re'&o"s#$ re*orted. P"n!hes %ro)

set o% r ts 9ere son&! ted &n


41/144

#$s te 9 s #o ded onto @6 3 !r$# )&de SDS ,e#0 e#e!tro*horesed &n Tr&s6,#$!&

"nder den t"r&n, !ond&t&ons nd tr ns%erred to n&tro!e##"#ose )e)7r ne B&

Her!"#es0 CA &n Tr&s6,#$!&ne 7"%%er 9&th 13Q 3 )eth no#. 4e)7r nes 9ere 7

%or 1 h t roo) te)*er t"re 9&th < 9t 'o# non6% t ): &n Tr&s67"%%ered s #

'o# 'o# T9een 3 TBS6T9een


42/144

A"st&n0 TX . RNA !on!entr t&on nd *"r&t$ 9 s deter)&ned "s&n, N n

N nodro*0 /)&n,ton0 DE . S )*#es $&e#d&n, #ess th n


43/144

! UANTIFICATION AND ANALYSIS OF PCR RESULTS

D t 9ere *#otted !ross !$!#es %or dete!ted %#"ores!en!e 7o'e the 7 !:,ro"n

s&,n #. The ( !oord&n te t the thresho#d o% )*#&%&! t&on 9 s des&,n ted the

s )e s"re o% )RNA +" nt&t t&on. To ' #&d te s )*#e '& 7&t$0 the s#o*e o%

#&ne t the &n%#e!t&on *o&nt %or e !h s )*#e 9 s !o)* red nd s )*#es 9&th s#o

de'& t&n, )ore th n < %ro) the )e n 9ere d&s! rded. St nd rd d"t&on e%%

!"r'es 9ere ,ener ted "s&n, *re'&o"s#$ deter)&ned *r&)ers. St nd rd !"r'es %or 7ot

,enes h d !orre# t&on !oe%%&!&ents ,re ter th n 3. 3. Inter !t&on tests 7et9een

d"r&n, )"#t&6*#e( 9ere ne, t&'e. The re# t&'e )RNA e(*ress&on o% GAPDH )RNA 9

"sed %or nor) #& t&on. D t 9ere n #$ ed 7$ the 6ZZCT )ethod .

E PERIMENTAL PROTOCOL

There 9ere e&,ht se* r te ,ro"*s "sed %or )et 7o#&s) ! ,e st"d&es5 dr", 'eh&

n ['e sh ) #&, ted n \ 0 dr", 'eh&!#e n ['e BDL n \ 11 0 'eh&!#e ICV &n%"se

#&, ted n \ 0 #os rt n ICV &n%"sed sh ) #&, ted n \ ? 0 'eh&!#e ICV &n%"s

13 0 #os rt n ICV &n%"sed BDL n \ 13 0 SC #os rt n sh ) #&, ted n \ 13 0 S

BDL n \ 13 . These n&) #s 9ere "sed %or *"n!h !o##e!t&on nd /estern 7#ot n #$

SFO AT1 R 7"nd n!e. So)e s )*#es 9ere not &n!#"ded &n the n #$s&s d"e to

*rote&n or )RNA re!o'er$.

43


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STATISTICAL ANALYSIS

A## res"#ts re *resented s )e ns ] SE4. D $ 9 ter0 "r&ne0 sod&")0 nd

)e s"re)ents 9ere n #$ ed "s&n, t9o69 $ re*e ted )e s"res ANOVA. Post6ho!

n #$s&s o% s&,n&%&! nt ) &n e%%e!ts 9 s ! rr&ed o"t "s&n, St"dent Ne9) nQ

ho! test S&,) *#ot0 S n -ose0 CA . Other d t 9ere n #$ ed 7$ one69 $ n #

' r& n!e. S&,n&%&! n!e 9 s set t P ^ 3.3


45/144

de!re sed 9e&,ht , &n &n BDL n&) #s T 7#e 10 *\3.32 sh ) 's. BDL . As res

)et 7o#&! d t 9ere nor) #& ed to 7od$ 9e&,ht.

BDL r ts dr n: s&,n&%&! nt#$ )ore 9 ter th n sh ) #&, ted !ontro#s F&,

*^3.3< 7"t h d no s&,n&%&! nt d&%%eren!es &n "r&ne 'o#")e F&,"re A . B

sho9ed ,re ter 9 ter 7 # n!e sh )5 .2 ] 1. )# :,61d $61 's. BDL5 > .? ] .? )# :,61

d $61 nd sod&") 7 # n!e sh )5 .? ] 3. )E+ :,61d $61 's. BDL5 @.1 ] 3.> )E+ :,61

d $61 !o)* red to sh ) #&, ted r ts. 4&n&*")* n ['e BDL r ts e(h&7&ted de!re sed r

sod&") e(!ret&on !o)* red to sh ) r ts F&,"re B0 *^3.331 .

AT1R *rote&n F&,"re >A nd )RNA F&,"re >B 7"nd n!es 9ere s&,n&%

&n!re sed P^3.3< &n the SFO o% BDL r ts !o)* red to sh ). /here s OVLT AT

*rote&n 7"nd n!e sho9ed s#&,ht &n!re sed e(*ress&on &n BDL r ts F&,"re @

there 9 s no !orres*ond&n, &n!re se &n AT1 R )RNA F&,"re @B . There 9

s&,n&%&! nt d&%%eren!e &n AT1R *rote&n or AT1 R )RNA 7"nd n!e &n 4nP

tre t)ent ,ro"*s F&,"re @C nd @D res*e!t&'e#$ .

E FFECTS OF CHRONIC LOSARTAN INFUSIONS ON FLUID BALANCE AND AT" RECEPTOR

E PRESSION IN BDL RATS

S&) r to )&n&*")* n ['e r ts0 *# s) os)o# #&t$ nd he) to!r&t 9ere #oBDL r ts th t re!e&'ed s #&ne or #os rt n &n%"s&on e&ther ICV or s"7!"t neo"s#$

Not 7#$0 ICV #os rt n tre t)ent d&d not %%e!t *# s) os)o# #&t$ &n BDL r ts

45


46/144

These o7ser' t&ons &nd&! te th t ICV #os rt n tre t)ent t th&s !on!entr t&on )

%%e!t ' so*ress&n re#e se or the res"#t&n, d"t&on # h$*on tre)& . F"rther)

#&, ted r ts &)*# nted 9&th s"7!"t neo"s #os rt n )&n&*")* , &ned s&,n&%&!

9e&,ht !o)* red to ## other tre t)ent ,ro"*s T 7#e 0 *^3.3< . As res"#t0

)et 7o#&! d t 9ere nor) #& ed to 7od$ 9e&,ht.

Tre t)ent 9&th ICV #os rt n *re'ented &n!re sed dr&n:&n, 7eh '&or e(h&7&

BDL r ts F&,"re < *^3.3< . BDL r ts th t re!e&'ed s"7!"t neo"s )&n&*")*s !ont

the s )e !on!entr t&on o% #os rt n s !ontro# %or *er&*her # !t&ons o% IC

#os rt n sho9ed no &nh&7&t&on o% dr&n:&n, 7eh '&or or !h n,e o% ren # sod&

These o7ser' t&ons &nd&! te th t the CNS 9 s the #&:e#$ s&te o% !t&on o% #os

d&d not o7t &n e'&den!e th t s"7!"t neo"s #os rt n )od&%&ed the d&se se

Interest&n,#$0 BDL r ts re!e&'&n, ICV &n%"s&on o% 3. s #&ne 'eh&!#e sho9

"r&ne o"t*"t !o)* red to sh ) n&) #s F&,"re < *oss&7#$ d"e to &n!re sed 9 ter nd * rt& # ' so*ress&n es! *e.

/e d&d not o7ser'e d&%%eren!e &n ren # sod&") e(!ret&on &n BDL r ts &n

or s"7!"t neo"s )&n&*")* tre t)ent ,ro"*s F&,"re ; . There 9 s #so no d&%%eren

%ood &nt :e 7et9een ## the tre t)ent ,ro"*s d t not sho9n .

Los rt n &n%"sed !hron&! ##$ &nto the # ter # 'entr&!#es *re'ented the &n

AT1R *rote&n e(*ress&on o7ser'ed &n dr", n ['e nd 'eh&!#e &n%"sed r ts F&,

/estern 7#ot n #$s&s o% AT1R e(*ress&on &n the OVLT F&,"re ?A nd 4nPO

46


47/144

?B o% BDL r ts tre ted 9&th ICV #os rt n d&d no9 sho9 s&,n&%&! nt d&%%

the tre t)ent ,ro"*s.

47


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Table 1: Measurements of serum osmolality, hematocrit, protein, liver weight to bodyweight ratio, and final body weight from sham (Sham) and bile duct ligated (BDL) rats.

nOsmolality(mOsm/kg)

Hematocrit(%)

PlasmaProtein

(g/dl)

Liver Wt/Body

Wt

FinalBody

Weight (g)

Sham 11 !" # " $ # 1 &' # !'1!'! #!'!! 470 9

B L 1 "* # 1+ $$ # 1+ &'$ # !'1!'!&1 #!'!!"+ 441 11

D t re )e n ] SE4. _*^3.3< !o)* red to sh ) #&, t&on.

48


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Table 2: Measurements of serum osmolality, hematocrit, protein, liver weight to bodyweight ratio and final body weight from sham (Sham) and bile duct ligated (BDL) ratsthat were treated with either vehicle ICV (Veh), losartan ICV (Los), or losartansubcutaneously (SCLos).

nOsmolality(mOsm/kg)

Hematocrit(%)

PlasmaProtein

(g/dl)

Liver Wt/Body

WtFinal BodyWeight (g)

Sham,eh 1! 303 3 46 1

7.4 0.2

0.029 0.004 437 10

ShamLos * 300 2 47 1

7.4 0.2

0.032 0.004 425 12

ShamS-Los 1! 304 1 49 1 7.7 0.2 0.032 0.004 482 8B L,eh 1! 293 2* 41 2*

7.6 0.2

0.066 0.004* 408 14

B LLos * 292 3* 42 1*

7.7 0.2

0.068 0.004* 411 8

B LS-Los 1! 297 1 46 1

7.5 0.2

0.075 0.004* 426 6

D t re )e n ] SE4. _*^3.3< !o)* red to ## sh ) #&, t&on `*^3.3< !o)* red

Sh ) SC Los.

49


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50

Figure 2: Average daily water intake and urine output(A) and average daily sodium intake and urinarysodium excretion (B) from Sham and BDL rats. Valuesare normalized to body weight (Kg) and represented asmeans SEM. *Statistically significant, P


51/144

51

Figure 3: Western Blot analysis of AT1Rabundance (A) and RT-qPCR analysis ofAT1aR mRNA (B) from brain punches

containing the SFO of Sham and BDL ratstaken 28 days after BDL surgery. The dataare presented as mean SEM relativeprotein and mRNA abundance. * Statistically significant, P


52/144

52

Figure 4: Western Blot analysis of AT1R abundance (top) and RT-qPCR analysis ofAT1aR mRNA (bottom) from brain punches containing the OVLT (A & B) and MnPO(C & D) of Sham and BDL rats taken 28 days after BDL surgery. The data are presentedas mean SEM relative protein and mRNA abundance. *Statistically significant, P


53/144

53

. v e r a g e

a

i l y

, o l m e

( m l

0 g

1 )

!

"!

$!

2!

%!

1!!

1"! H" O 3ntake4rine **

*

*

Figure 5: Average daily water intake and urine output normalized to body weight(Kg) from Sham and BDL rats treated with either vehicle (Veh), losartan (Los),or sub-cutaneous losartan (SCLos). Values are means SEM. *Statisticallysignificant, P


54/144

54

Figure 6: Average daily sodium intake and renal sodium excretion from Sham andBDL rats treated with either vehicle (Veh), losartan (Los), or sub-cutaneous losartan(SCLos). Values are means SEM. Animal numbers are 8-10 for each group. Nosignificant differences between groups.


55/144

55

Figure 7: Western Blot analysis of AT1R abundance from brain punches

containing the SFO of Sham and BDL rats from each treatment group taken 28days after BDL surgery. *Statistically significant, P


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56

Figure 8: Western Blot analysis of AT1R expression in brainpunches of the A) OVLT, and B) MnPO of Sham and BDL ratstreated with either vehicle or Losartan ICV taken 28 days afterBDL surgery. Numbers within the bars indicate the number ofanimals.


57/144

DISCUSSION

The ) &n %&nd&n,s o% th&s st"d$ re th t 1 9 ter &nt :e &n!re sed &n B

BDL &s sso!& ted 9&th &n!re sed SFO AT1R *rote&n nd AT1 R )RNA0 nd >

o% the AT1R nt ,on&st #os rt n &nto the 'entr&!#es 7#o!:ed the &n!re sed 9 ter &

&n!re sed 7"nd n!e o% AT1R *rote&n &n the SFO. A !oro## r$ %&nd&n, &s

os)o# #&t$ 9 s not %%e!ted 7$ ICV #os rt n &n%"s&on 9h&!h s",,est th

n,&otens&n s&,n #&n, ) $ not *# $ ro#e &n BDL sso!& ted &n **ro*r& te

re#e se. T :en to,ether0 these res"#ts &nd&! te th t SFO AT1 R e(*ress&on ) $ *# $

&n the re,"# t&on o% dr&n:&n, 7eh '&or 7"t not the !t&' t&on o% SON ne"rose!re

&n the !onte(t o% BDL !&rrhos&s.

In *re'&o"s st"d$0 o"r # 7 *ro'&ded ,ro"* o% BDL r ts 9&th s #&ne to

to de!re se PRA 7$ &n!re s&n, sod&") de#&'er$ to the d&st # t"7"#e o% the :&dne$1;2. Th&s

) n&*"# t&on 9 s sso!& ted 9&th s&,n&%&! nt de!re se &n PRA nd' so*ress&n !o)* red to BDL r ts *ro'&ded on#$ 9&th 9 ter1;2. These %&nd&n, *ro'&ded

!orre# t&on # e'&den!e th t the ren&n6 n,&otens&n s$ste) ) $ !ontr&7"te to &n

*# s) ' so*ress&n !on!entr t&ons &n BDL r ts. E r#&er &n '&'o st"d&es sho

str"!t"res !o)*r&s&n, the # )&n ter)&n #&s !ont &n %"n!t&on # ren&n6 n,&ote

th t e(tends &nto the SON 1;?61210 nd &n '&tro e#e!tro*h$s&o#o,&! # st"d&es h 'e #so

th t An, II ! n !t *ost6s$n *t&! ##$ to !t&' te non6s*e!&%&! ! t&on&! !ond

de*o# r& e SON ne"rons nd *re6s$n *t&! ##$ to % !&t te ,#"t ) te re#e s

! n !ross the 7#ood67r &n 7 rr&er t s"%%&!&ent#$ h&,h !on!entr t&ons to 7#o!:57


58/144

II 7&nd&n,1?;0 ho9e'er 9e d&d not test re!e*tor 7&nd&n, %ter #os rt n d)&n&st

There%ore 9e ! nnot *ro'&de d&re!t &n%or) t&on 7o"t the n to)&! # s*e!&%&

o7ser'ed e%%e!ts. C&r!"# t&n, An, II ) $ *# $ ro#e &n the !t&' t&n, !&r!")'ent

or, n %%erents to the SON ho9e'er0 the *resent d t &nd&! te th t !entr # n,

s&,n #&n, &s not ne!ess r$ %or d"t&on # h$*on tre)& d"r&n, BDL

S"7!"t neo"s #os rt n 9 s "sed to !ontro# %or #e : ,e %ro) ICV #os rt n &nt

*er&*her # !&r!"# t&on. T9o *re'&o"s st"d&es sho9ed th t #os rt n ! n #ter t

d&se se *ro!ess 7$ *re'ent&n, the *ro,ress&on o% #&'er %&7ros&s0 *ort # h$*erte

h$*erd$n )&! !&r!"# t&on . Th&s st"d$ "sed !on!entr t&on o% Los rt n th t 9

s) ##er th n the )&n&)") dose th t &s re+"&red to red"!e !&rrhos&s or h$*erd$n

!&r!"# t&on. There%ore0 red"!t&on &n dr&n:&n, 7eh '&or nd tten" t&on o% &n

o7ser'ed &n BDL r ts &s d"e to the !t&on o% #os rt n &n the CNS.

St"d&es o% dr&n:&n, 7eh '&or &n BDL r ts h 'e re !hed )&(ed !on!#"s&on%&nd&n, th t BDL r ts !ons")e )ore 9 ter &s !ons&stent 9&th *re'&o"s st"d$ o% r

9ere not ho"sed &n )et 7o#&! ! ,es1;2. Ho9e'er0 nother st"d$ sho9ed no d&%%eren!e

7et9een sh ) #&, ted nd BDL r t dr&n:&n, 7eh '&ors12>. In th&s # tter st"d$0 the r ts

9ere ho"sed &n )et 7o#&! ! ,es %or ; d $s 9&tho"t *er&od o% !!#&) t&on d"r&

t&)e the r ts 9ere ,&'en or # doses o% 9 ter 7$ , str&! , ' ,e s !ontro# %or #os

dr", d)&n&str t&on. The "se o% , str&! , ' ,e ) $ h 'e &n%#"en!ed 7 se#&ne 9 te

d"e to stress or the 'o#")e o% %#"&d de#&'ered d&re!t#$ to the , stro&ntest&n # tr !t

58


59/144

Other st"d&es h 'e sho9n th t BDL r ts h 'e &n!re sed sod&") **et&te0 7"t

not %&nd e'&den!e o% &n!re sed 9 ter &nt :e . It h s #so 7een re*orted th t the &

sod&") &nt :e &s &nde*endent o% the ren&n6 n,&otens&n s$ste) . The d&%%eren

these o7ser' t&ons ) $ 7e d"e to the d&%%erent r t str &n "sed &n the *re'&o"s 9or:

h s 7een sho9n to h 'e d&%%erent d&*so,en&! res*ons&'eness to os)ot&! or 7#ood *

!h ##en,es1? .

/he n&) #s nor) ##$ re,"# te *# s) os)o# #&t$ d"r&n, !"te os)o

!h ##en,es thro",h d&%%erent& # ' so*ress&n re#e se to #ter ren # %ree 9 ter

dr&n:&n, 7eh '&or &s nother 9 $ th t n&) #s ! n re,"# te *# s) os)o# #&t$

*h$s&o#o,&! # !onte(t o% *ro,ress&'e#$ de!re s&n, *# s) os)o# #&t$ sso!& ted

!&rrhos&s0 &n!re sed 9 ter &nt :e0 #on, 9&th &n **ro*r& te ' so*ress&n0 !o"#

to h$*on tre)& . T9o !&r!")'entr&!"# r or, ns0 the SFO nd OVLT re &n'o#'ed &n

!entr # !ontro# o% %#"&d &nt :e re# ted to !h n,es &n *# s) An, II nd ) $ * rt&e#e' ted 9 ter &nt :e &n BDL r ts. Ho9e'er0 the e%%e!t o% Los rt n on dr&n:&n,

) $ not 7e s*e!&%&! to SFO AT1Rs 9h&!h 9ere "*re,"# ted &n BDL r ts0 nd there%

*re!&se re,&ons nd )o#e!"# r )e!h n&s)s res*ons&7#e %or &n!re sed 9 ter &nt :e

re) &n to 7e deter)&ned.

Fro) o"r n #$s&s o% AT1R e(*ress&on &n the # )&n ter)&n #&s0 9e !on!#BDL &s sso!& ted 9&th n &n!re sed 7"nd n!e o% AT1 R )RNA nd AT1R *ro

the SFO o% BDL r ts !o)* red to sh ) !ontro#s. These d t re s&) r to other st"

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th t sho9ed &n!re sed 7"nd n!e o% AT1R &n the SFO o% n&) # )ode#s th t h d

*er&*her # ren&n6 n,&otens&n s$ste) !t&' t&on . Nor) ##$ re!e*tors do9n6re,"#

*resented 9&th n 7"nd n!e o% #&, nd. Ho9e'er0 &n res*onse to e#e' ted !&r!"# t

II0 AT1Rs * r do(&! ##$ &n!re se &n 7"nd n!e. Th&s *heno)enon 9 s %&rst o7

st"d&es o% r ts de*r&'ed o% 9 ter1@


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ICV d)&n&str t&on o% An, II *otent#$ st&)"# tes dr&n:&n, &n r ts0 nd t

**e rs to 7e 7oth ne!ess r$ nd s"%%&!&ent %or d&*so,en&! e%%e!t1>261>. Other st"d&es

"t& &n, tr ns,en&! )&!e h 'e sho9n th t the &n!re sed *rod"!t&on o% SFO

&n!re sed 9 ter &nt :e1@30 nd th t !hron&! 7r &n6restr&!ted o'ere(*ress&on o% AT

res"#ts &n h&,her 7 se#&ne 9 ter &nt :e nd n &n!re sed 9 ter &nt :e res

e(o,eno"s ICV An, II 1@1. St"d&es o% s*ont neo"s#$ h$*ertens&'e r ts 0 r ts de*r&'e

9 ter 0 nd r ts 9&th !on,est&'e he rt % "re h 'e #so sho9n &n!re sed e(*res

AT1R &n the SFO0 9h&!h &s sso!& ted 9&th &n!re sed 9 ter &nt :e 9here )e

T :en to,ether0 these d t s",,est n &)*ort nt ro#e %or !entr # An, II nd es*e!&

AT1R &n the re,"# t&on o% 9 ter &nt :e 7$ SFO ne"rons *ro e!t&n, to the 4n

A#tho",h the ro#e o% SFO n,&otens&n s&,n #&n, &n st&)"# t&n, dr&n:&n, 7eh '&

9e## des!r&7ed0 !h n,es th t o!!"r &n th&s s$ste) d"r&n, !&rrhos&s h 'e not

*re'&o"s#$ re*orted.

Western blot and RT-qPCR analysis of OVLT and MnPO AT1R protein and

mRNA abundance failed to show consistent differences between sham and BDL groups

indicating that increased expression of AT1R in the lamina terminalis of BDL rats is

limited to SFO. There might be an increase in Angiotensin type 1b (AT1b) receptor

subtype, which would be detected in our western blot results due to the lack of specificity

of the antibody; however, AT1aR mRNA in the SFO was also increased. Therefore, it is

likely that the increased AT1R protein abundance in the SFO reflects increased

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expression of AT1aR. Both AT1a and AT1b receptor subtypes have unique regulatory

functions 336 3; however both subtypes also share 95% homology in mRNA and amino

acid sequences, and have similar Ang II binding profiles 3>. It is possible that the

increased drinking behavior we observed may have been due to increased activation of

AT1b receptors. Davisson et al 3@ showed that brain AT1b receptors are important

mediators of drinking behavior due to central administration of Ang II. However, it was

also shown that central AT1aR overexpression increased both baseline and Ang II-

induced drinking behavior 1@1. Both of these transgenic studies were performed in mice,

and it is possible that the drinking behavior of rats may respond differently to AT1aR

knockdown or silencing. Therefore, future studies that selectively target AT1a and/or

AT1b receptor subtypes in the SFO during BDL, such as with viral-mediated shRNA

knockdown microinjected into the SFO 3


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&nh&7&t&on o% !entr # n,&otens&n re!e*tors &s not s"%%&!&ent to re'erse

de'e#o*)ent o% d"t&on # h$*on tre)& *rod"!ed 7$ BDL. Nonethe#ess0 these %

do he#* &dent&%$ *otent& # ro#e %or SFO AT1Rs &n e(!ess dr&n:&n, d"r&n, #

Better "nderst nd&n, o% ho9 9 ter &nt :e nd ' so*ress&n re#e se re re,"# ted

os)ot&! ##$ nd non6os)ot&! ##$ !o"#d &n%#"en!e tre t)ent *roto!o#s %or d&

h$*on tre)& .

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CHAPTER >5 ANGIOTENSIN II RECEPTOR SUBTYPE 1A AT1AR GENEKNOCKDO/N IN NEURONS OF THE SUBFORNICAL ORGAN SFO

PREVENTS INCREASED DRINKING BEHAVIOR IN BILE DUCT LIGATEDRATS.

A77re'& ted t&t#e5Authors: -ose*h D / #!h10 0 nd -. Tho) s C"nn&n,h )1

1De* rt)ent o% Inte,r t&'e Ph$s&o#o,$0 nd C rd&o' s!"# r Rese r!h Inst&t"teUn&'ers&t$ o% North Te( s He #th S!&en!e Centre t Fort /orth0> F#o$d C"r# Dr&'eS n Anton&o0 TX 2?

K#$ %or&s: ' so*ress&n0 AT1R0 dr&n:&n, 7eh '&or0 7e d"!t #&, t&on

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ABSTRACT

Be d"!t #&, t&on BDL ! "ses !on,est&'e #&'er % "re th t

he)od$n )&! !h n,es res"#t&n, &n d"t&on # h$*on tre)& *oss&7#$ d"e to &

9 ter &nt :e nd ' so*ress&n re#e se. Th&s *ro e!t tested the h$*othes&s th t n,

s&,n #&n, thro",h the s"7%orn&! # or, n SFO ",)ents dr&n:&n, 7eh '&or &n B

A ,enet&! ##$ )od&%&ed deno6 sso!& ted '&r"s !ont &n&n, shRNA %or n

re!e*tor s"7t$*e 1 AT1 R ,ene 9 s )&!ro&n e!ted &nto the s"7%orn&! # or, n

r ts to :no!:do9n e(*ress&on. T9o 9ee:s # ter0 BDL or sh ) s"r,er$ 9 s *er%or)ed.

R ts 9ere ho"sed &n )et 7o#&! !h )7ers %or )e s"re)ent o% %#"&d nd %ood &nt

"r&ne o"t*"t. The r ts 9ere e"th n& ed ? d $s %ter BDL s"r,er$ %or n #$s&s. A

r ts 9 s *er%"sed %or &))"noh&sto!he)&! # n #$s&s nd se!ond ,ro"* 9 s "s

# ser6! *t"re )&!rod&sse!t&on %or n #$s&s o% SFO AT1 Rs ,ene e(*ress&on.

sho9ed &n!re sed 9 ter &nt :e th t 9 s tten" ted &n r ts th t re!e&'ed

)&!ro&n e!t&on o% AT1 R shRNA R4 T9o69 $ ANOVA %o##o9ed 7$ SNK *o

*^3.3


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% !tors &nd"!e the d"t&on # h$*on tre)& sso!& ted 9&th th&s d&se

st&)"# t&n, the re#e se o% ' so*ress&n des*&te *ro,ress&'e *# s) h$*on tr

In!re sed !&r!"# t&n, n,&otens&n II An, II &s one % !tor th t !o"#d !ontr

s$)* tho6e(!&t t&on0 &n!re sed AVP re#e se nd e#e' ted %#"&d &nt :e 7$ !t&

!&r!")'entr&!"# r or, ns .

The # )&n ter)&n #&s !ont &ns t9o !&r!")'entr&!"# r or, ns5 the or,

' s!"#os") o% the # )&n ter)&n #&s OVLT nd the s"7%orn&! # or, n SFO

9&th&n these "n&+"e %ore7r &n re,&ons # !: %"n!t&on # 7#ood 7r &n 7 rr&er

these str"!t"res to 7e sens&t&'e to !&r!"# t&n, % !tors s"!h s *# s) os)o# #&t$ nd

12


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MATERIALS AND METHODS

ANIMALS

E(*er&)ents 9ere !ond"!ted on d"#t ) #e S*r ,"e6D 9#e$ r ts C en'&ron)ent "nder 1 1

!$!#e 9&th #&,ht onset t 3233h. R ts h dad libitum !!ess to %ood nd 9 ter e(!e*t

d"r&n, the s"r,&! # *ro!ed"res. A## e(*er&)ent # *ro!ed"res 9ere !ond"!ted &

!!ord n!e 9&th the ,"&de#&nes o% the P"7#&! He #th Ser'&!e nd 9ere **ro'ed

Un&'ers&t$ o% North Te( s He #th S!&en!e Centre Inst&t"t&on # An&) # C re

Co))&ttee.

BILE DUCT LIGATION MODEL OF HEPATIC CIRRHOSIS

E !h n&) # 9 s nesthet& ed 9&th &so%#"r ne &n o($,en . A%

nesthet& ed0 the 7do)en 9 s sh 'ed nd the re 9 s !#e ned 9&th &od&ne nt&

)&d#&ne &n!&s&on 9 s *er%or)ed on the 7do)en nd the !o))on 7e d"!t 9 s &s

7$ 7#"nt d&sse!t&on nd !"t 7et9een t9o #&, t"res. E !h n&) # 9 s then ret"rned

! ,e nd )on&tored d $ "nt "sed %or the e(*er&)ent. V&s" # &ns*e!t&on o%

&n the *er&tone # ! '&t$ 9 s *er%or)ed d $ %ter s"r,er$. An$ r t sho9&n, )o

e.,.0 % "re to thr&'e0 s&,n&%&! nt de!re se &n %ood or 9 ter &nt :e 9 s e

th&o7"t 7 r7&t # In !t&n 0 S&,) 0 St. Lo"&s0 4O 133 ), :, &.*. . Sh ) #&, te

9ere s"7 e!ted to the s )e s"r,&! # *ro!ed"re 9&th the e(!e*t&on th t the 7e d"!t 969


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P LASMA MEASUREMENTS

Tr"n: 7#ood 9 s !o##e!ted &nto 1.< )# )&!ro!entr&%",e t"7e. T9o he* r&

!ont &n&n, ! *# r$ t"7es 9ere %#ed 9&th 7#ood %ro) th&s s )*#e %or

he) to!r&t. The rest o% the s )*#e 9 s !entr&%",ed < )&n 130333 , nd

s )*#e o% ser") 9 s re)o'ed %or )e s"r&n, os)o# #&t$ "s&n, ' *or *ress"r

os)o)eter /es!or In!. Lo, n0 UT 0 nd sod&") !on!entr t&on "s&n, %#

*hoto)eter -en9 $ PFP20 V/R Intern t&on #0 R dnor0 PA . Ser") *rote&n

)e s"red 7$ re%r !to)eter$ n t&on # Proto)eter0 N t&on # Instr")ents0 B #t&)ore0

'F OS B IMMUNOHISTOCHEMISTRY

Se* r te ,ro"*s o% sh ) #&, ted AAV6GFP6SC4 &n e!ted n \ 0 sh ) #&

AAV6GFP6sh6AT1 &n e!ted n \ 13 0 BDL AAV6GFP6SC4 &n e!ted n \ 2 0

AAV6GFP6sh6AT1 &n e!ted n \ ? r ts 9ere "sed &n &))"noh&sto!he)&str$ st"d&

deter)&ne the e%%e!ts o% SFO AT1 re!e*tor :no!:do9n nd BDL on bFosB st &n&

the SON. Fo"r 9ee:s ? d $s %ter BDL or sh ) #&, t&on s"r,er$0 r ts

nesthet& ed 9&th th&o7"t 7 r7&t # In !t&n0 133 ), :, &.*. nd *er%"sed tr

9&th 336@33 )# o% @ * r %or) #deh$de &

&))"noh&sto!he)&str$ s *re'&o"s#$ des!r&7ed1


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15 don:e$ nt&6,"&ne *&, C$> nd Don:e$ nt&6)o"se C$ 0

- !:son I))"noRese r!h0 /est Gro'e0 PA .

C ONFOCAL IMAGING AND !UANTIFICATION OF IMMUNOHISTOCHEMISTRY

Se!t&ons 9ere e( )&ned "s&n, n O#$)*"s IX6 DSU !on%o! # )&!ros!o

e+"&**ed %or e*&%#"ores!en!e 9&th the **ro*r& te e(!&t t&on e)&ss&on %

&) ,&n, bFosB0 AVP nd OXY *os&t&'e !e##s &n the SON. I) ,es 9ere ! *t"red "s&

Ret&, 6SRV ! )er c6&) ,&n,0 S"rre$0 Br&t&sh Co#")7& 0 C n d . Br &n

&dent&%&ed "s&n, P (&nos nd / tson1?1. Se!t&ons !ont &n&n, the SON #o! ted 63.?3 )) to

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61.?3 )) *oster&or to 7re,) 9ere n #$ ed %or bFosB0 AVP nd OXY !e## n")7ers

!o#o! #& t&on.

L ASER CAPTURE MICRODISSECTION

A se* r te set o% n&) #s !o)*r&sed o% the s )e %o"r tre t)ent ,ro"*s 9 s "

%or the # ser ! *t"re )&!rod&sse!t&on st"d&es0 n \ < %or ## %o"r ,ro"*s . E

nesthet& ed 9&th In !t&n 133 ), :, &.*. nd +"&!:#$ de! *&t ted ? d $s %

sh ) #&, t&on s"r,er$. Br &ns 9ere re)o'ed nd &))ed& te#$ sn * %ro en &n d

!oo#ed &so*ent ne. Ser& # !oron # se!t&ons 13 ) thro",h SFO 9ere !"t "s&n, !r

C41


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! UANTIFICATION AND ANALYSIS OF PCR RESULTS

D t 9ere *#otted !ross !$!#es %or dete!ted %#"ores!en!e 7o'e the 7 !:,ro"n

s&,n #. The ( !oord&n te t the thresho#d o% )*#&%&! t&on 9 s des&,n ted the

s )e s"re o% )RNA +" nt&t t&on. To ' #&d te s )*#e '& 7&t$0 the s#o*e

t n,ent& # to the &n%#e!t&on *o&nt %or e !h s )*#e 9 s !o)* red nd s )*#es 9&

' #"es de'& t&n, )ore th n < %ro) the )e n 9ere d&s! rded. The re# t&'e e(*ress&o

GAPDH )RNA 9 s "sed %or nor) #& t&on. D t 9ere n #$ ed 7$ the 6ZZCT )ethod.

E PERIMENTAL PROTOCOL

There 9ere %o"r se* r te r ndo)#$ ##o! ted ,ro"*s "sed %or )et 7o#&s) !

st"d&es5 AAV6GFP6SC4 sh ) #&, ted n \ 1 AAV6GFP6SC4 BDL n \ 1

GFP6sh6AT1 sh ) #&, ted n \ 1? 0 nd AAV6GFP6sh6AT1 BDL n \ 1; . Stereo

s"r,er$ 9 s *er%or)ed t9o 9ee:s *r&or to sh ) #&, t&on or BDL s"r,er$ to ##o9 %

re!o'er$. R ts 9ere )o'ed &nto )et 7o#&! ! ,es %or )e s"re)ent o% d $ %ood &n

9 ter &nt :e0 nd "r&ne o"t*"t on the 1?th d $ %ter sh ) #&, t&on or BDL s"r,er$

des!r&7ed 7o'e 4et 7o#&! C ,es . At the end o% the e(*er&)ent0 e !h r t

nesthet& ed 9&th In !t&n 133 ), :, &.*. nd *er%"sed %or &))"noh&sto!he)&str

)# 7#ood s )*#e 9 s t :en 7$ ! rd& ! *"n!t"re 7e%ore e !h *er%"s&on %or )e s

*# s) os)o# #&t$0 sod&")0 he) to!r&t0 nd *# s) *rote&n s des!r&7ed 7o'e. E

#&'er 9 s re)o'ed nd 9e&,hed s des!r&7ed 7o'e. The 7r &n %ro) e !h r t 9

*ro!essed %or ' so*ress&n nd o($to!&n &))"noh&sto!he)&str$ "s&n, C$>6 nd

!on ", ted se!ond r$ nt&7od$ res*e!t&'e#$ s des!r&7ed 7o'e. Se!t&ons !ont &n75


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SFO 9ere n #$ ed %or GFP "s&n, n e*&%#"ores!en!e )&!ros!o*$ to deter)&ne #

o% the &n e!t&on s&tes nd tr nsd"!t&on o% SFO ne"rons. A se* r te set o% r ts %

the %o"r ,ro"*s n\


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RESULTS

C HARACTERI(ATION OF BDL E PERIMENTAL MODEL AND PLASMA MEASUREMENTS

P# s) s )*#es %ro) sh ) !ontro#s nd BDL r ts 9ere "sed to )e s"re *# s)

os)o# #&t$0 ser") sod&")0 nd he) to!r&t. A## BDL r ts o% e !h tre t)ent ,

e(h&7&ted s&,n&%&! nt#$ #o9er *# s) os)o# #&t$ *^3.31 !o)* red to sh

n&) #s o% the s )e ,ro"* T 7#e @ . Not 7#$0 AAV6AT1 R6shRNA )&!ro&n e

the SFO % ed to %%e!t *# s) os)o# #&t$ &n BDL r ts. Th&s o7ser' t&on &

SFO AT1 R s&,n #&n, ) $ not 7e &n'o#'ed &n the *"t t&'e &n **ro*r& te re#

' so*ress&n or the !on!o)&t nt d"t&on # h$*on tre)& th t 9e o7ser'e &n BD

S&) r#$0 *# s) he) to!r&t 9 s #so s&,n&%&! nt#$ de!re sed &n BDL !o)*

#&, ted r ts *^3.310 's. ## sh ) ,ro"*s . Ch n,es &n *# s) os)o# #&t$ nd he)

re not d"e to !h n,es &n *rote&n s s",,ested 7$ the % "re to dete!t d&%%er

*# s) *rote&n )e s"re)ents. Co)* red to sh )0 BDL r ts th t !o)*#eted the

e(*er&)ent d&s*# $ed "nd&!e 9&th e#e' ted ser") 7&r"7&n0 he* tos*#eno)e

de'e#o*ed !&rrhot&! #&'er s e' #" ted 7$ ,ross '&s" # e( )&n t&on nd s&,n

&n!re sed #&'er69e&,ht6to67od$69e&,ht r t&os *^3.331 . The res"#ts de)onstr te e

&nd"!t&on o% #&'er % "re nd d"t&on # h$*on tre)& &n BDL r ts th t 9e

7$ &n e!t&on o% the AAV6AT1 R shRNA tr nsd"!t&on 'e!tor.

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AT" AR SH RNA VIRAL TRANSDUCTION AND GENE KNOCKDOWN

S"!!ess%"# '&r # de#&'er$ o% the AAV6GFP6shRNA ,ene e(*ress&on ! sset

'er&%&ed 7$ !on%o! # )&!ros!o*$ &n ## n&) #s F&,"re . AAV6shRNA 'e!tor

,reen %#"ores!ent *rote&n GFP th t %"n!t&oned s ) r:er to &nd&! te th

de#&'er$ nd !e##s %%e!ted. S&( 9ee:s %ter d&re!t SFO )&!ro&n e!t&on o% A

!ontro# nd AT1 R 'e!tors0 9e o7ser'ed ro7"st GFP e(*ress&on &n the SFO F&,"re

GFP *os&t&'e !e##s 9ere #so o7ser'ed &n the re s"rro"nd&n, the SFO0 not 7#$ d

the SFO nd o!! s&on ##$ #on, the &n e!tor tr !:s. A## r ts &n!#"ded &n t

!ont &ned GFP # 7e#&n, &n the SFO. Se'en n&) #s 9ere e(!#"ded %ro) the st"d$ d

&n e!t&on s&tes th t 9ere too dors # to *rod"!e GFP # 7e#&n, &n the SFO. To

e%%e!t&'eness o% AAV6AT1 R6shRNA tr nsd"!t&on to :no!:do9n AT1 R e(*ress&

) 7r &n s#&!e se!t&ons !ont &n&n, the SFO 9ere "sed %or # ser )&!rod&sse!t&

SFO nd n #$s&s o% AT1 R )RNA 7"nd n!e 7$ RT6+PCR. AT1 R )RNA 7"nd

9 s s&,n&%&! nt#$ &n!re sed &n the SFO o% BDL r ts tre ted 9&th the !ontr

!onstr"!t !o)* red to sh ) #&, ted n&) #s &n e!ted 9&th e&ther the AAV6SC4 or A

AT1 R shRNA '&r # tr nsd"!t&on 'e!tors F&,"re 13 *^3.31 . SFOs %ro) BDL n

&n e!ted 9&th the AT1 R6shRNA !ont &ned s&,n&%&! nt#$ #ess AT1 R )RNA

!o)* red to BDL n&) #s re!e&'&n, the !ontro# AAV6SC4 shRNA F&,"re 13 *\3.3

BDL SC4 's. BDL sh6AT1 . Th"s0 )&!ro&n e!t&on o% the AAV6AT1 R sh

tr nsd"!t&on 'e!tor e%%e!t&'e#$ *re'ented the &n!re sed AT1 R )RNA e(*ress

o7ser'ed &n BDL r ts.

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F LUID INTAKE AND RENAL SODIUM E CRETION

There 9 s no s&,n&%&! nt !h n,e &n dr&n:&n, 7eh '&or or ren # sod&") e

7et9een sh ) n&) #s tre ted 9&th e&ther the !ontro# SC4 shRNA or the AT1 R shRN

/e o7ser'ed s&,n&%&! nt &n!re se &n 'er ,e d $ 9 ter !ons")*t&on o% B

&n e!ted 9&th the !ontro# shRNA !o)* red to sh ) #&, ted n&) #s tre ted 9&th

!ontro# nd AT1 R shRNA tr nsd"!t&on 'e!tors F&,"re 11 *^3.3


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!onse+"ent#$ &n sod&") &nt :e F&,"re 1 . Both BDL n&) # ,ro"*s #so sho9

&n!re sed sod&") 7 # n!e F&,"re 1> BDL shAT1 5 *^3.3< !o)* red to 7oth s

,ro"*s BDL SC45 *^3.3< !o)* red to sh ) SC4 .

'F OS B IN THE SON OF BDL RATS

BDL &n!re sed bFosB st &n&n, nd !o#o! #& t&on o% bFosB 9&th ' so

the SON F&,"re 1@ . The n")7ers o% bFosB *os&t&'e !e##s &n the SON 9 s s&,n&

&n!re sed @ 9ee:s %ter BDL &n 7oth '&r # tre t)ent ,ro"*s !o)* red to 7oth sh

#&, ted !ontro#s F&,"res 1@ = 1< . F"rther)ore0 9e o7ser'ed &n!re sed n")7er

Walch Phddissert

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